BACKGROUND Plasmodium falciparum deficient for hrp2 and hrp3 genes are a threat to malaria management and elimination, since they escape widely used HRP2-based rapid diagnostic tests and treatment. Hrp2/hrp3 deletions are increasingly reported from all malaria endemic regions but are currently only identified by laborious methodologies. METHODS We developed a novel hydrolysis probe-based, quantitative, real-time PCR (4plex qPCR) for detection and discrimination of P. falciparum infection (cytb) and hrp2 and hrp3 gene status, and to control assay validity (btub). A cross-sectional, diagnostic accuracy study was performed in Gabon for assay validation and deletion screening. FINDINGS In parallel to identification of P. falciparum infection in samples down to 0.05 parasites/µl, the 4plex qPCR enabled specific and valid interrogation of the parasites´s hrp2 and hrp3 genes in one go - even in low parasitemic samples. The assay was precise and robust also when performed in a routine healthcare setting in Gabon. The risk of falsely identifying hrp2 or hrp3 deletion was reduced by 100-fold compared to conventional PCR. Evaluation against microscopy was performed on 200 blood samples collected in Gabon: sensitivity and specificity of 4plex qPCR (cytb) were 100% and 80%, respectively. Stringent testing revealed hrp2 deletion in 2 of 95 P. falciparum positive and validated samples. INTERPRETATION The novel 4plex qPCR is sensitive, accurate and allows resource-efficient rapid screening. Monitoring and mapping of hrp2/hrp3 deletions is required to identify areas where control strategies may need to be adapted to ensure appropriate patient care and ultimately achieve malaria elimination. FUNDING BMBF (03VP00402).

中文翻译：

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通过新颖的高通量检测恶性疟原虫hrp2和hrp3缺失来监测疟疾快速诊断测试的潜在威胁：一项横断面，诊断准确性研究。

背景技术缺乏hrp2和hrp3基因的恶性疟原虫对疟疾的管理和消除构成威胁，因为它们逃避了广泛使用的基于HRP2的快速诊断测试和治疗。从所有疟疾流行地区越来越多地报道了Hrp2 / hrp3缺失，但目前仅通过费力的方法来鉴定。方法我们开发了一种新型的基于水解探针的定量实时PCR（4plex qPCR），用于检测和鉴别恶性疟原虫感染（cytb）以及hrp2和hrp3基因状态，并控制测定的有效性（btub）。在加蓬进行了横断面，诊断准确性研究，用于分析验证和缺失筛选。研究结果与鉴定低至0.05寄生虫/ µl的样品中的恶性疟原虫感染同时，4plex qPCR可以一次又一次地对寄生虫的hrp2和hrp3基因进行特异性和有效的询问-即使在低寄生虫样品中也是如此。在加蓬的常规医疗机构中进行测定时，该测定方法也精确且可靠。与常规PCR相比，错误鉴定hrp2或hrp3缺失的风险降低了100倍。对在加蓬收集的200个血液样本进行了显微镜评估：4plex qPCR（cytb）的敏感性和特异性分别为100％和80％。严格测试显示，在95例恶性疟原虫阳性和验证样本中，有2例hrp2缺失。解释新颖的4plex qPCR灵敏，准确，可进行资源高效的快速筛选。需要监测和映射hrp2 / hrp3缺失，以识别可能需要调整控制策略以确保适当的患者护理并最终实现消除疟疾的区域。资金BMBF（03VP00402）。